All dogs included in this study were from a rescue shelter in southern Italy (40° 0′ 45ʺ North, 18° 9′ 38ʺ East, Casarano, Apulia region). This area is endemic for CanL [18] and is a suitable environment for different Phlebotomus spp. (i.e., Phlebotomus perniciosus, Phlebotomus perfiliewi, and Phlebotomus neglectus), which transmit L. infantum [19]. In this geographical area, the sand fly season lasts from late May to late October, with two density peaks during July and August [19].
In September 2021, during the transmission season (T1), shelter dogs were physically examined, a clinical sign-based score for CanL, ranging from 0 (absence of clinical signs) to 19 (severely sick), was assigned [20], and a blood sampling was performed for laboratory tests to complete the dog’s health status.
Briefly, blood samples were collected from either the cephalic or jugular veins and placed in a K3 ethylenediaminetetraacetic acid (EDTA) tube (2 ml) to undergo routine hematology (i.e., complete blood count [CBC] with reticulocyte count), ESR, and modified Knott’s test. Exactly 5 ml of blood were placed in a plain tube to obtain serum after centrifugation (15 min at 1500 × g) to perform a complete biochemical panel, including acute-phase proteins (i.e., CRP and ferritin), serum capillary electrophoresis, and serology.
The erythrocyte sedimentation rate was measured using a point-of-care device (MINIPET, DIESSE, Diagnostica Senese S.p.A., Siena, Italy), and the reference interval was established as 0–10 mm/h [21]. Results from CBC (Siemens, ADVIA 2120), serum biochemical analysis (Beckman Coulter, Clinical Chemistry Analyzer AU680), and electrophoresis (SEBIA, Capillarys 2 Flex Piercing) were achieved with the same methods in all tested samples.
Serum samples were analyzed for L. infantum antibodies by IFAT as previously described [22]. They were considered positive if clear cytoplasmic and membrane fluorescence of L. infantum promastigotes from a cut-off dilution of 1:80 was evident. Positive sera were titrated by serial dilutions (i.e., 1:80, 1:160, 1:320, 1:640, 1:1280) up to 1:2560. Samples were considered negative if they failed to produce a positive result at a 1:80 dilution. All serological tests were read in a double-masked manner by two different operators.
Serum samples were also tested for Anaplasma phagocytophilum (MegaCor Diagnostik, Horbranz, Austria), and Ehrlichia canis (Biopronix Agrolabo, Scarmagno, Italy) antibodies by IFAT, and for the snail-borne pathogen Angiostrongylus vasorum diagnosed by the ELISA technique [23, 24].
Moreover, blood samples were processed using a modified Knott’s test for the detection and identification of circulating microfilariae (mfs) of Dirofilaria spp. as previously described [25]. Then, dogs without microfilariae were tested for Dirofilaria immitis antigen by ELISA (Filarcheck, Agrolabo, Scarmagno, Italy).
According to the laboratory results, dogs were considered finally eligible for this study at T1 if they were: (1) seropositive to L. infantum; (2) negative to E. canis, A. phagocytophilum, Dirofilaria spp., and A. vasorum; (3) not treated with antileishmanial drugs in the previous 6 months; and (4) not treated with insecticides or repellents in the previous 6 months.
In January 2022 (nontransmission season; T2), all dogs included underwent clinical examination, laboratory analyses, and serological testing as described above. During the study period, enrolled dogs did not receive any antileishmanial drug, as well as insecticides or repellents. Treatment decisions were indeed under the responsibility of the shelter’s attending veterinarian, in accordance with management policies and available financial resources. The researchers’ role was limited to clinical monitoring using the previously detailed diagnostic tools.
The normality of the results was checked using the Kolmogorov–Smirnov test. Laboratory parameters (i.e., albumin, albumin/globulin ratio, CRP, ESR, ferritin, gamma globulins, globulins, hematocrit, total iron, and total proteins) were normally distributed and reported as mean and standard deviation (M ± SD), and as frequencies and percentages (%) for categorical variables. The Wilcoxon matched-pairs signed-rank test was used to compare the differences between pairs of observations in the groups at time points (T1 and T2) for continuous variables. When testing the null hypothesis of no association, the probability level of error at two tails was 0.05. All the statistical computations were made using StataCorp. 2021. Stata Statistical Software: Release 17. College Station, TX, USA: StataCorp LLC.